The development of memory T cells is essential to protection against many intracellular pathogens. Our studies have demonstrated that during an immune response to infection, a large portion of the effector and memory antigen-specific CD8 T cells are located in the non-lymphoid tissues. Memory cells residing in lymphoid and non-lymphoid tissues exhibit distinct functional and phenotypic characteristics. These subsets can be characterized as central memory T cells (Tcm, residing in lymphoid tissue) and effector memory T cells (Tem, found predominantly in non-lymphoid tissues). Our hypothesis proposes that nave T cell precursor frequency and antigen concentration and duration control memory lineage development. This proposal will focus on determining the role of these factors in driving memory lineage choice with respect to phenotype, function and location. The specific aims are: Aim 1. To determine whether antigen availability during primary and secondary responses to infection controls memory T cell development. Whether the duration of antigen availability and the timing of T cell entry into the response affect memory development will be tested using a novel system in which MHC-restricted antibody blockade will be used to inhibit CD4 and CD8 T cell responses following infection. Aim 2. To determine the effects of endogenous naove CD8 T cell frequency and infection type on memory development. Our preliminary data indicated that endogenous naove CD8 T cells could be isolated and quantitated. Moreover, the naove CD8 T cell frequency and the infection type affected memory CD8 T cell development. Based on these data, the studies in this aim will test the hypothesis that endogenous naove CD8 T cell frequencies in conjunction with the duration of antigen availability control memory lineage choice. Aim 3. To determine the effect of naove precursor frequency, infection type and antigen availability on in situ T cell activation . Our preliminary results identify formation of discrete clusters of antigen-specific CD8 T cells early during the primary immune response to infection. In addition, during reactivation, memory CD8 T cells migrate from the splenic marginal zones and in fact the B cell areas to the T cell zones in the white pulp. Using confocal microscopy and transgenic mice expressing a fluorescent reporter in dendritic cells we will determine the effect of endogenous naove precursor frequency, antigen duration and infection type on primary and secondary CD8 T cell responses in situ. In addition, we will visualize the location and reactivation characteristics of Tcm and Tem cells. These studies will provide a valuable link between anatomical characteristics of T cell activation and the functional development of memory T cells. Overall, these studies will provide important insight into the control of memory T cell lineage development in vivo in response to infection.Project narrative. This proposal is focused on understanding the generation of memory T cells in response to infection. Memory T cells are essential to provide protection against many infections so this research has substantial impact on understanding immunity and vaccine design.